Cutting edge: Immunosuppressant as adjuvant for tolerogenic immunization

Abstract

Vaccination for autoimmune and alloimmune diseases has long been an attractive idea. Yet, there is no suitable adjuvant to forcefully steer the immune response toward tolerance. In this study we show that dexamethasone, a potent glucocorticoid immunosuppressant, can function as a tolerogenic adjuvant when applied together with peptide immunogen. BALB/c mice with pre-established delayed-type hypersensitivity to hen OVA were immunized with an OVA-derived, MHC II-restricted peptide (OVA(323-339)) in the presence of dexamethasone. The treatment caused long-term desensitization in treated animals to hen OVA via a dexamethasone-dependent tolerogenic mechanism that blocks maturation of dendritic cells and expands OVA(323-339)-specific CD4(+)CD25(+)Foxp3(+) regulatory T cells in vivo. Similar treatment of NOD mice using dexamethasone and an insulin-derived, MHC II-restricted peptide (B:9-23) prevented predisposed spontaneous diabetes. Remarkably, in both models, dexamethasone-augmented immunization induced long-term persistent, Ag-specific regulatory T cells responsive to recall Ags. These results reveal for the first time the potential usefulness of immunosuppressants as tolerogenic adjuvants.

FIGURE 1

Suppressed immunization induces immune tolerance against established DTH. A, BALB/c mice with pre-established DTH were divided into four groups and each was treated with an indicated combination of DEX and OVA_323–339. All groups were then retested for DTH at a footpad. B, The test and negative control groups were tested again 4 –5 mo later. C, Treg (CD4⁺CD25⁺Foxp3⁺) in blood samples, taken immediately before (open bar) and 48 h after (filled bar) the second DTH test, were quantified relatively to total CD4⁺ cells. D, The same blood taken at 48 h from non-treated mice (indicated as “Control”) or mice treated with DEX and peptide (“Test”) was further analyzed for the presence of OVA_323–339-specific Treg. White cells were labeled with CFSE, stimulated in culture with OVA_323–339, and analyzed for CFSE dilution (cell division) by flow cytometry, gating on CD4⁺CD25⁺Foxp3⁺ cells (Treg). The observed cell division was Ag dependent because no CFSE dilution was seen in the absence of OVA_323–339 (data not shown). Bar, mean and SD of two or three independent experiments, each using six mice per group (n & 6); *, p < 0.05 between the indicated pair.

FIGURE 2

Suppressed immunization blocks DC maturation and preferentially expands Ag-specific Treg. A, DO11.10 mice were divided into four groups, each injected at a hind footpad with the indicated combination of DEX and OVA_323–339. Three days later, draining LN (popliteal) were recovered and analyzed by flow cytometry, gating on CD11c⁺ cells (total DC). Immature (CD83⁻CD86^low) and mature (CD83⁺CD86^high) DC were quantified relatively to total DC. B, IL-10-producing (IL-10⁺) DCs were quantified relatively to total DC. C, Activated Teff (CD4⁺CD25⁺Foxp3⁻) and Treg (CD4⁺CD25⁺Foxp3⁺) in same LN were quantified relatively to total CD4⁺ T cells. D, CFSE-labeled DO11.10 CD4⁺ T cells were coinjected with OVA_323–339 into BALB/c mice that had been either pretreated (indicated as “DEX + peptide”) or non-pretreated (“peptide”) with DEX. As a negative control, the T cells were also injected without OVA_323–339 into non-pretreated mice (“PBS”). Four days later, total cells from draining LN were stained for KJ1–26, CD25, and Foxp3 and analyzed by flow cytometry, gating on KJ1–26⁺/CD25⁺ cells. Proliferating Treg are shown in the upper (Foxp3⁺) left quadrant and proliferating Teff in the lower (Foxp3⁻) left quadrant. E, Treg from DO11.10 mice treated with DEX and OVA_323–339 were cocultured with Teff (CD4⁺CD25⁻) from naive DO11.10 mice, along with syngeneic accessory cells and OVA_323–339. Proliferation was assessed by [³H]thymidine incorporation. Treg¹ and Treg² denote Treg from immunized and nonimmunized DO11.10 mice, respectively. Bar, mean and SD from 2–4 independent experiments, each using at least two mice per group (n ≥ 2); *, p < 0.05 between the indicated pair.

FIGURE 3

Suppressed immunization protects animals from predisposed autoimmune diseases. A, Prediabetic NOD mice (n = 6 per group) were treated with PBS (open circle), DEX alone (triangle), B:9–23 alone (square), or both DEX and B:9–23 (filled circle). Mice were checked weekly for glycosuria. Shown is one of two independent experiments with similar results. The difference between the “DEX and B:9–23” and “DEX alone” or “B:9–23 alone” groups is statistically significant (p < 0.002). B, Mice that were treated with DEX and B:9–23 and subsequently remained diabetes-free at 61 wk of age were rechallenged with B:9–23. Splenic CD4⁺ T cells were isolated 5 day later, labeled with CFSE, and stimulated in culture with B:9–23 (indicated as “Test”) or OVA_323–339 (“Control Ag”). Control splenic T cells were obtained from nonimmunized prediabetic NOD mice that had been similarly rechallenged with B:9–23. The control cells were stimulated in culture with B:9–23 (“Control”). CFSE dilution was analyzed 3 days later by flow cytometry, gating on CD4⁺CD25⁺ cells (Treg and activated Teff). Shown are quadrant plots separating Treg (Foxp3⁺), Teff (Foxp3⁻), Ag-specific (CFSE-low), or non-antigen-specific (CFSE-high) cells. The percentage of cells in each quadrant is indicated. Shown is one of two independent experiments with similar results. C, Prediabetic NOD mice were treated with PBS (indicated as “Control”) or DEX and B:9–23 (“Test”), as described in A. At the completion of the treatment, blood samples were taken from both groups. White cells were labeled with CFSE and stimulated with B:9–23 in culture. As control for Ag specificity, an aliquot of white cells from the test mice were stimulated with OVA_323–339 (“Control Ag”). Cells were analyzed by flow cytometry as described in B. Ag-specific cells were identified by CFSE dilution and quantified relatively to total CD4⁺CD25⁺ cells gated. Bar, mean and SD of two independent experiments using two or three mice per group; *, p < 0.0016 between the indicated pair.